The present invention relates to recombinant DNA molecules comprising the promoter region of the sel-12 gene of Caenorhabditis elegans (C. elegans) or promoter regions of genes homologous to the sel-12 gene, being capable of conferring expression of a heterologous DNA sequence in neural cells, advantageously all neural cells, preferably at all stages of development. The present invention also provides vectors comprising the DNA molecules. The present invention also relates to pharmaceutical and diagnostic compositions comprising such recombinant DNA molecules and vectors. Furthermore, the present invention relates to transgenic animals, e.g., in species ranging from the fruit fly to mammals, such as non-human animals, comprising the aforementioned recombinant DNA molecules or vectors stably integrated into their genome, as well as their use for the identification of substances capable of complementing a neuronal disorder. The present invention also relates to the use of the inventive recombinant DNA molecules and vectors for the preparation of pharmaceutical compositions for treating, preventing, and/or delaying a neuronal disorder in a subject. Furthermore, the recombinant DNA molecules and vectors of the invention also can be used for the preparation of pharmaceutical compositions for inducing a neuronal disorder in a non-human animal.
Several documents are cited throughout the text of this specification with a References section provided prior to the claims. Citations can be in the form of a numerical designation or designations in brackets, with the numerical designation or designations corresponding to a document or documents similarly numbered in the Reference section. Each of the documents cited herein (including any manufacturer""s specifications, instructions, etc.) are hereby incorporated herein by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.
In the field of neuroscience and medical therapy, there is a great demand for test systems to study the function and interaction of gene products, the malfunction or expression of which cause neuronal diseases. Such systems would also be suitable for drug development against neuronal diseases. A prominent example for a neuronal disease is familial Alzheimer""s disease (FAD). The majority of cases with FAD are linked to mutations of the presenilin (PS) genes. These genes are homologous to the sel-12 gene of C. elegans, which has been postulated to function in the facilitated signaling by lin-12 and glp-1 [15].
Of the genes involved in FAD, mutations within the PS genes are most frequently associated with the early onset of the disease [1]. These genes encode two highly homologous proteins (PS-1, PS-2) [1-5] which appear to adopt a seven to nine transmembrane (TM) domain structure [1-5]. Most of the mutations which co-segregate with FAD patients occur within the PS-1 gene whereas only two mutations have been identified within the PS-2 gene so far [1-5]. Both PS proteins are predominantly located within the endoplasmic reticulum and the early Golgi [6-8], where they co-localize with the xcex2-amyloid precursor protein (xcex2APP) [7]. Mutations in the PS genes cause elevated levels of the long form of amyloid xcex2-peptide terminating at amino acid 42/43 [5, 9, 10], the key player in sporadic Alzheimer""s disease (AD) as well as in FAD [11]. It was shown recently that PS-1 is proteolytically cleaved [12, 13] within the large loop between TM6 and TM7[13]. Since endogenous full-length PS proteins were not detected [7, 12, 13], it was suggested that the proteolytic fragments of PS proteins exhibit their biological function. This is further supported by the finding that the FAD associates PS-1 xcex94exon9 mutant [14] which lacks the cleavage site, accumulated as full-length protein [12].
The biological functions of the PS proteins are yet unknown. However, sequence similarities to the C. elegans sel-12 [15] (42%) and to a lesser extent to the SPC-4 [1, 16] proteins suggest a functional conservation of these proteins. The conservation of topology of sel-12 and PS-1 further supports this assumption [17, 18]. Interestingly, the FAD linked mutations within the human PS genes all occur at positions which are conserved with the two homologous C. elegans genes [1-5, 15, 16] suggesting that these mutations are located at functionally important positions. Mutations in sel-12 suppress the Multivulva phenotype of an hyper-active lin-12/Notch mutant. sel-12 mutants themselves have a strong egg-laying (Egl) defect which is reminiscent of the Egl phenotype caused by reducing lin-12 activity [15]. These results suggested that sel-12 is either directly involved in lin-12 signaling or in the receptor transport or recycling [15].
Recently, Levitan [20] developed a model system to study PS structure and function in C. elegans. In their system, PS-1 was expressed from an engineered lin-12 promoter variant described in Wilkinson and Greenwald [23]. This promoter is expressed in the vulva precursor cells, most likely the only cells where sel-12 function is required for the correct execution of egg-laying behavior. It was shown that PS-1 expression in the vulva precursor cells can functionally replace sel-12. For studying all aspects of genes involved in neuronal diseases such as FAD, in particular their effect on the psycho/motoric system however, the system described by Levitan [20] suffers from several drawbacks.
For example, although the PS genes in humans are ubiquitously expressed [2, 3], their neural expression is a prerequisite for understanding their role in the pathophysiology of FAD [6]. The promoter employed in Levitan [20], however, is not expressed in most neural cells, and, therefore, defects of sel-12 mutants like an uncoordinated (kinker) phenotype and a general lethargy of the animals cannot be complemented by their promoter constructs. Furthermore, the lin-12 promoter only functions efficiently in the genetic background of smg-1 mutants [23]. However, smg-1 mutants significantly suppress the sel-12 phenotype [20].
Thus, a technical problem present in the art and believed not heretofore provided is providing promoter regions capable of conferring expression of a heterologous DNA sequence in all neural cells, preferably at all stages of development.
Since Alzheimer is a disease of the nervous system, the inventor decided to study the function and interaction of proteins involved in this disease in neural cells since otherwise non-informative or even false positive results may be obtained.
Thus, an object of the present invention can be to provide promoter regions capable of conferring the expression of a heterologous DNA sequence in neural cells, advantageously all neural cells, and preferably at all stages of development. Accordingly, it is an object of the invention to address, and preferably provide a solution, to the technical problem in the art, by providing embodiments of the invention.
Further objects of the invention can include providing, inter alia:
an isolated nucleic acid molecule, e.g., a nucleotide sequence such as an isolated DNA molecule or a recombinant DNA molecule comprising the promoter of the sel-12 gene from C. elegans or the promoter region of a gene homologous to the sel-12 gene being-capable of conferring expression in neural cells, advantageously all neural cells, and preferably at all stages of development, and optionally operatively linked thereto at least one isolated nucleic acid molecule or nucleotide-sequence, e.g., a heterologous DNA sequence;
a vector comprising at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule;
a pharmaceutical composition comprising at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule and/or vector, and optionally a pharmaceutically acceptable carrier;
a diagnostic composition comprising at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule and/or vector, and optionally suitable means for detection;
a transgenic non-human animal comprising stably integrated into its genome at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule and/or vector;
a method for the identification of a chemical and/or biological substance capable of complementing a neuronal disorder comprising contacting an inventive transgenic non-human animal (preferably having a neuronal disorder, such as from containing at least one inventive isolated nucleic acid molecule) with a plurality of compounds, and determining those compounds which are capable of complementing the neuronal disorder of the transgenic non-human animal;
a method for the identification of a chemical and/or biological substance capable of interfering Presenilin/APP interaction comprising: contacting an inventive transgenic non-human animal (preferably having Presenilin/APP interaction, such as from containing at least one inventive isolated nucleic acid molecule) with a plurality of compounds, and determining those compounds which are capable of interfering with Presenilin/APP interaction; and
use of at least one inventive transgenic non-human animal for identifying a substance capable of complementing a neuronal disorder or a method for identifying a substance capable of complementing a neuronal disorder (the use or method can comprise administering a substance to an inventive transgenic non-human animal and determining whether the substance complements the physical and/or behavioral disorder displayed by the transgenic non-human animal as a result of a mutant sel-12 gene or gene which is homologous to the sel-12 gene).
Accordingly, the present invention provides an isolated nucleic acid molecule, e.g., an isolated or recombinant DNA molecule, comprising:
(a) the promoter region of the sel-12 gene from C. elegans or a promoter region of a gene homologous to the-sel-12 gene being capable of conferring expression in neural cells, advantageously substantially all neural cells, preferably at substantially all stages of development; and optionally
(b) operatively linked thereto at least one heterologous nucleic acid molecule, e.g., an isolated nucleic acid molecule such as DNA, for instance such a nucleic acid molecule comprising a coding sequence.
Further embodiments of the invention can include, inter alia:
an isolated nucleic acid molecule, e.g., a nucleotide sequence such as an isolated DNA molecule or a recombinant DNA molecule comprising the promoter of the sel-12 gene from C. elegans or the promoter region of a gene homologous to the sel-12 gene being capable of conferring expression in neural cells, advantageously all neural cells, and preferably at all stages of development, and operatively linked thereto at least one isolated nucleic acid molecule or nucleotide sequence, e.g., a heterologous DNA sequence;
a vector comprising at least one inventive isolated nucleic acid molecule6 e.g., recombinant DNA molecule;
a pharmaceutical composition comprising at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule and/or vector, and optionally a pharmaceutically acceptable carrier;
a diagnostic composition comprising at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule and/or vector, and optionally suitable means for detection;
a transgenic non-human animal comprising stably integrated into its genome at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule and/or vector;
a method for the identification of a chemical and/or biological substance capable of complementing a neuronal disorder comprising contacting an inventive transgenic non-human animal (preferably having a neuronal disorder, such as from containing at least one inventive isolated nucleic acid molecule) with a plurality of compounds, and determining those compounds which are capable of complementing the neuronal disorder of the transgenic non-human animal;
a method for the identification of a chemical and/or biological substance capable of interfering Presenilin/APP interaction comprising: contacting an inventive transgenic non-human animal (preferably having Presenilin/APP interaction, such as from containing at least one inventive isolated nucleic acid molecule) with a plurality of compounds, and determining those compounds which are capable of interfering with Presenilin/APP interaction; and
use of at least one inventive transgenic non-human animal for identifying a substance capable of complementing a neuronal disorder or a method for identifying a substance capable of complementing a neuronal disorder (the use or method can comprise administering a substance to an inventive transgenic non-human animal and determining whether the substance complements the physical and/or behavioral disorder displayed by the transgenic non-human animal as a result of a mutant sel-12 gene or gene which is homologous to the sel-12 gene).
The promoter region can comprises the nucleotide sequence as depicted in SEQ ID NO: 1 (FIG. 3) part(s) thereof which capable of conferring expression in all neural cells at all stages of development.
The exogenous or heterologous DNA sequence can encode a protein or functional part thereof involved in neuronal development and/or diseases, for instance a protein involved in Alzheimer disease, e.g., Presenilin or a functional part thereof, or a protein which is functionally equivalent to Presenilin or a part thereof. The protein can be encoded by a DNA sequence selected from the group consisting of
(a) DNA sequences comprising a nucleotide sequence encoding the sequence as given in SEQ ID NO: 3 (FIG. 5);
(b) DNA sequences comprising the nucleotide sequence as given in SEQ ID NO: 2 (FIG. 4);
(c) DNA sequences comprising a nucleotide sequence which hybridizes with a complementary strand of a nucleotide sequence of (a) or (b); and
(d) DNA sequences comprising a nucleotide sequence which is degenerate to a nucleotide sequence of (c).
The exogenous or heterologous DNA sequence can encode the amyloid precursor protein (APP) or its proteolytic product (Axcex2), a functional part thereof or a protein functional equivalent to APP or Axcex2 or any mutated versions thereof. The exogenous or heterologous DNA sequence can be selected from the group consisting of
DNA sequences comprising a nucleotide sequence encoding the amino acid sequence as given in SEQ ID NO: 5 (FIG. 7);
(b) DNA sequences comprising the nucleotide sequence as given in SEQ ID NO: 4 (FIG. 6);
(c) DNA sequences comprising a nucleotide sequence which hybridizes with a complementary strand of a nucleotide sequence of (a) or (b); and
(d) DNA sequences comprising a nucleotide sequence which is degenerate to a nucleotide sequence of (c).
The exogenous or heterologous DNA sequence can encode a mutated version of a protein which has lost its capability of complementation of a sel-12 mutant phenotype.
The vector can include one or more of the inventive isolated nucleic acid molecules, e.g., recombinant DNA molecules, or one or more exogenous or heterologous isolated nucleic acid molecules, e.g., exogenous or heterologous DNA sequences.
For instance, an inventive vector can contain at least one exogenous or heterologous DNA sequence encoding: (i) a protein or functional part thereof involved in neuronal development and/or diseases, for instance a protein involved in Alzheimer disease, e.g., Presenilin or a functional part thereof, or a protein which is functionally equivalent to Presenilin or a part thereof, for instance, the protein can be encoded by a DNA sequence selected from the group consisting of
(a) DNA sequences comprising a nucleotide sequence encoding the sequence as given in SEQ ID NO: 3 (FIG. 5);
(b) DNA sequences comprising the nucleotide sequence as given in SEQ ID NO: 2 (FIG. 4);
(c) DNA sequences comprising a nucleotide sequence which hybridizes with a complementary strand of a nucleotide sequence of (a) or (b); and
(d) DNA sequences comprising a nucleotide sequence which is degenerate to a nucleotide sequence of (c); and (ii) the amyloid precursor protein (APP) or its proteolytic product (Axcex2), a functional part thereof or a protein functional equivalent to APP or Axcex2 or any mutated versions thereof, e.g., the exogenous or heterologous DNA sequence can be selected from the group consisting of
(a) DNA sequences comprising a nucleotide sequence encoding the amino acid sequence as given in SEQ ID NO: 5 (FIG. 7);
(b) DNA sequences comprising the nucleotide sequence as given in SEQ ID NO: 4 (FIG. 6);
(c) DNA sequences comprising a nucleotide sequence which hybridizes with a complementary strand of a nucleotide sequence of (a) or (b); and
(d) DNA sequences comprising a nucleotide sequence which is degenerate to a nucleotide sequence of (c). And, the inventive vector can contain an inventive promoter region for each exogenous or heterologous DNA sequence of (i) and (ii), or a promoter region for both (i) and (ii), i.e., an inventive promoter may drive expression of more than one exogenous or heterologous DNA sequence; and if desired, this can be exploited in any suitable arrangement in an inventive vector.
The vector can further comprise a selectable marker, preferably pha-1.
The invention also comprehends a cell transformed with at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule or at least one vector; and, the cell can be a prokaryotic or a eukaryotic cell. And, the cell can be in an animal, such as a non-human animal, such that the invention comprehends transgenic animals.
The inventive transgenic non-human animal can have a neuronal disorder. The neuronal disorder can be caused by a mutant sel-12 gene or a gene which is homologous to the sel-12 gene. The neuronal disorder-can caused by the expression of at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule, or at least one inventive vector. The animal, e.g., transgenic non-human animal, can be any desired species, for instance, from fruit fly to mammal, e.g., any suitable invertebrate or vertebrate, such as a nematode, e.g., C. elegans, or a mammal, e.g., a laboratory animal such as felines (such as domesticated cats or kittens), canines (such as domesticated dogs or puppies), rodents (such as mice, rabbits, gerbils, rats and the like), ruminants (such as sheep, goats and bovines and the like), primates (monkeys, apes and the like), inter alia.
In the aforementioned inventive methods or uses the chemical or biological substance can be selected from the group consisting of peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, neurotransmitters, peptidomimics and PNAs; and, the neuronal disorder can be Alzheimer disease.
The invention further comprehends the use of at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule, for inducing a neuronal disorder in a non-human. animal; for instance, the use of at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule, for the preparation of a pharmaceutical composition for inducing a neuronal disorder in a non-human animal. In these embodiments the exogenous or heterologous DNA sequence can encode a mutated version of a protein which has lost its capability of complementation of a sel-12 mutant phenotype. The neuronal disorder can be uncoordinated behavior and/or lethargic movement. And, the animal can be any desired species, for instance, from fruit fly to mammal, e.g., any suitable invertebrate or vertebrate, such as a nematode, e.g., C. elegans, or a mammal, e.g., a laboratory animal as discussed above.
In addition, since the inventive promoter region can confer expression in neural cells, advantageously all neural cells, and preferably at all stages of development, the at least one isolated nucleic acid molecule or nucleotide sequence, e.g., a heterologous DNA sequence (operatively linked to the promoter region) can encode a beneficial biological product such that the inventive combination of the promoter region and isolated nucleic acid molecule can be employed for numerous treatments or therapies, e.g., for treatment or therapy of neuronal disorders (e.g., uncoordinated behavior, lethargic movement, Alzheimer disease, inter alia); for instance, for the introduction into human or animal individuals or populations of missing genes or parts thereof, or of genetic material for modification, replacement or repair of defective genes.
The invention thus comprehends vectors, cells and tranogenic animals, such as mammals (including humans) containing such an inventive promoter region and isolated nucleic acid molecule combination, as well as compositions containing such an inventive combination or vectors or cells for delivery to an animal for purposes of such treatments or therapies, and treatment or therapeutic methods employing the inventive combination or vectors or cells comprising administering such to an individual in need of such treatment or therapy; and, the animal can be pre-natal or post-natal, e.g., a treatment or therapy can be in an individual after birth or in a mature individual or prior to birth. This type of therapy or treatment can be effected by any suitable vector; and by gene therapy one can introduce the inventive combination in vivo into cells using a retroviral vector (Naldini et al., Science 272 (1996), 263-267; Mulligan, Science 260 (1993), 926-932) or another appropriate vector. In genetic diseases the introduction of a normal or a functionally adequate gene or portion thereof represents gene replacement therapy, which can be applicable to numerous disorders, such as recessive disorders. A pharmaceutical composition for such treatment or therapy can be administered to the mammal at a suitable dose, which can be determined from this disclosure and knowledge in the art, without undue experimentation by the skilled artisan taking into consideration typical factors such as the species, age, sex, weight, condition and genetics of the mammal, the route of administration, whether the treatment is pre-natally or post-natally, toxicity, inter alia.
Furthermore, in view of the foregoing, in vitro, use of the inventive promoter region, especially in combination with at least one isolated nucleic acid molecule or nucleotide sequence, e.g., a heterologous DNA sequence (operatively linked to the promoter region) may also be within the invention. Such in vitro uses can be to have in vitro expression of gene products from neural cells for subsequent use for administration to an animal, e.g., for treatment or therapy (e.g., of neuronal disorders), or for creating neural cells expressing gene products for subsequent infusion or reinfusion into an animal (ex vivo treatments or therapies, e.g., of neuronal disorders); and, the invention thus may also comprehend compositions and methods employing such in vitro expressed gene products or such in vitro created cells (e.g., a pharmaceutical composition comprising such gene products or cells, or a method for treating a neuronal disorder comprising administering the gene products, cells, or composition containing such).
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.